The present invention relates to novel compounds or pharmaceutically acceptable salts thereof, processes for preparing them, pharmaceutical compositions containing them and their use in therapy. The novel compounds referred to are positive modulators of nicotinic receptor agonists, said positive modulator having the capability to increase the efficacy of the said nicotinic receptor agonists.
Cholinergic receptors normally bind the endogenous neurotransmitter acetylcholine (ACh), thereby triggering the opening of ion channels. ACh receptors in the mammalian central nervous system can be divided into muscarinic (mAChR) and nicotinic (nAChR) subtypes based on the agonist activities of muscarine and nicotine, respectively. The nicotinic acetylcholine receptors are ligand-gated ion-channels containing five subunits (for reviews, see Colquhon et al. (1997) Advances in Pharmacology 39, 191-220; Williams et al. (1994) Drug News and Perspectives 7, 205-223; Doherty et al. (1995) Annual reports in Medicinal Chemistry 30, 41-50). Members of the nAChR gene family have been divided into two groups based on their sequences; members of one group are considered xcex2 subunits, while a second group are classified as xcex1 subunits (for reviews, see Karlin and Akabas (1995) Neuron 15, 1231-1244; Sargent (1993) Annu. Rev. Neurosci. 16, 403-443). Three of the xcex1 subunits, xcex17, xcex18 and xcex19, form functional receptors when expressed alone and thus presumably form homooligorneric receptors.
An allosteric transition state model of the nAChR involves at least a resting state, an activated state and a xe2x80x9cdesensitizedxe2x80x9d closed channel state (Williams et al., supra; Karlin and Akabas, supra). Different nAChR ligands can thus differentially stabilize the conformational state to which they preferentially bind. For example, the agonists ACh and (xe2x88x92)-nicotine stabilize the active and desensitized states.
Changes of the activity of nicotinic receptors has been implicated in a number of diseases. Some of these, e.g. myasthenia gravis and ADNFLE (autosomal dominant nocturnal front lobe epilepsy) (Kuryatov et al. (1997) J. Neurosci. 17(23):9035-47), are associated with reductions in the activity of nicotinic transmission either through a decrease in receptor number or increased desensitization, a process by which receptors become insensitive to the agonist. Reductions in nicotinic receptors have also been hypothesized to mediate cognitive deficits seen in diseases such as Alzheimer""s disease and schizophrenia (Williams et al., supra). The effects of nicotine from tobacco are also mediated by nicotinic receptors. Increased activity of nicotinic receptors may reduce the desire to smoke.
The use of compounds which bind nicotinic acetylcholine receptors in the treatment of a range of disorders involving reduced cholinergic function such as Alzheimer""s disease, cognitive or attention disorders, attention deficit hyperactivity disorders, anxiety, depression, smoking cessation, neuroprotection, schizophrenia, analgesia, Tourette""s syndrome, and Parkinson""s disease has been discussed in McDonald et al. (1995) xe2x80x9cNicotinic Acetylcholine Receptors: Molecular Biology, Chemistry and Pharmacologyxe2x80x9d, Chapter 5 in Annual Reports in Medicinal Chemistry, vol. 30, pp. 41-50, Academic Press Inc., San Diego, Calif.; and in Williams et al., supra.
However, treatment with nicotinic receptor agonists which act at the same site as ACh is problematic because ACh not only activates, but also blocks receptor activity through processes which include desensitization (for a review, see Ochoa et al. (1989) Cellular and Molecular Neurobiology 9, 141-178) and uncompetitive blockade (open-channel block); Forman and Miller (1988) Biophysical Journal 54(1):149-58. Furthermore, prolonged activation appears to induce a long-lasting inactivation. Therefore agonists of ACh can be expected to reduce activity as well as enhance it. At nicotinic receptors in general, and, of particular note, at the xcex17-nicotinic receptor, desensitization limits the duration of current during agonist application.
It has surprisingly been found that certain compounds can enhance the efficacy of agonists at nicotinic receptors. It is believed that compounds having this type of action (hereinafter referred to as xe2x80x9cpositive modulatorsxe2x80x9d) will be particularly useful for treatment of conditions associated with reductions in nicotinic transmission. In a therapeutic setting such compounds could restore normal interneuronal communication without affecting the temporal profile of activation. In addition, they would not produce long-term inactivation as prolonged application of agonist may.
According to the invention it has been found that compounds of Formula I: 
wherein,
R1a, R1b, R3a and R3b independently represent hydrogen, or C1-C4 alkyl;
R2a and R2b independently represents hydrogen, C1-C4 alkyl, or CH2CN;
A1 represents oxygen, sulfur, or NR4a;
B1 represents oxygen, sulfur, or NR4b;
R4a represents hydrogen, C1-C4 alkyl, C2-C4 alkenyl; or together R3a and R4a form a ring;
R4b represents hydrogen, C1-C4 alkyl, C2-C4 alkenyl; or together R3b and R4b form a ring;
A2 and B2 independently represent C(O), C(NH), OC(O), NHC(O), NHC(S), SO2, or a bond;
Z represents (CH2)nY(CH2)m;
n and m are independently 0-4;
Y represents oxygen, sulfur, NR5, CHR6, Ar, or Ccy;
Ccy represents a 5-10 membered carbocycle including cyclopentane and adamantane;
Ar represents phenyl, naphthyl; or a 5- or 6-membered heterocyclic ring containing zero to four nitrogens, zero to one sulfurs and zero to one oxygens;
Ar is optionally substituted with one or more substituents selected from: hydrogen, halogen,
C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, CN, NO2, CF3, CR7, NR8R9, and COOR10;
R7, R8 and R9 are independently hydrogen, C1-C4 alkyl, aryl, heteroaryl, C(O)R11, C(O)R12,
SO2R13, or, R8 and R9 together may be (CH2)jQ(CH2)k, where
Q is oxygen, sulfur, NR14, or a bond;
j is 2-4;
k is 0-2;
R5, R6, R10, R11, R12, R13 and R14 are independently hydrogen, C1-C4 alkyl, aryl, or heteroaryl;
or an enantiomer thereof, and pharmaceutically acceptable salts thereof, enhance the efficacy of agonists at nicotinic receptors.
Preferred compounds of the invention include the following:
1,3-Bis(indolyl-5-oxymethyl)benzene;
N,Nxe2x80x2-Di(5-indolyl)-1,3-adamantanedicarboxamide;
or an enantiomer thereof, and pharmaceutically acceptable salts thereof.
Unless otherwise indicated, the C1-C4 alkyl groups referred to herein, e.g., methyl, ethyl, n-propyl, n-butyl, i-propyl, i-butyl, t-butyl, s-butyl, may be straight-chained or branched, and the C3-C4 alkyl groups may also be cyclic, e.g., cyclopropyl, cyclobutyl.
Unless otherwise indicated, the C2-C4 alkenyl groups referred to herein may contain one or two double bonds, e.g., ethenyl, i-propenyl, n-butenyl, i-butenyl, allyl, 1,3-butadienyl. Unless otherwise indicated, the C2-C4 alkynyl groups referred to herein contain one triple bond, e.g., ethynyl, propynyl, 1-or 2-butynyl.
Halogen referred to herein may be fluoride, chloride, bromide, or iodide.
The compounds of the invention have the advantage that they may be less toxic, be more efficaous, be longer acting, have a broader range of activity, be more potent, produce fewer side effects, are more easily absorbed or have other useful pharmacological properties.
In the reaction schemes and text that follow, R1a, R1b, R2a, R2b, R3a, R3b, A1, A2, B1, B2, and Z, unless otherwise indicated, are as defined above for formula I. The compounds of formula I may be prepared according to the methods outlined in Schemes I and II.
Compounds of formula I may be prepared from compounds of formula IIIa or IIIb, wherein R1a, R1b, R2a, R2b, R3a, R3b, A1, A2, B1 and B2 are as defined in formula I, by reaction with an intermediate of formula IIa or IIb, wherein La or Lb is a suitable leaving group representing halogen, triflate (TfO), methanesulfonate (MsO), or p-toluenesulfonate (pTsO) and R1a, R1b R2a, R2b, R3a, R3b, A1, A2, B1, B2, and Z are as defined in formula I, in the presence of a suitable base and solvent. Intermediates of formula IIa or IIb may be prepared from compounds of formula IIIa or IIIb, wherein R1a, R1b, R2a, R2b, R3a, R3b, A1, and B1 are as defined in formula I, by selective reaction of one of the leaving groups of a compound of formula IV, wherein La and Lb are suitable leaving groups independently representing halogen, triflate (TfO), methanesulfonate (MsO), or p-toluenesulfonate (pTsO) and A2, B2 and Z are as defined in formula I, in the presence of a suitable base and solvent. Suitable bases include sodium carbonate (Na2CO3), cesium carbonate (Cs2CO3), potassium carbonate (K2CO3), triethylamine (TEA) or N,N-diisopropylethylamine (DIPEA). Suitable solvents for the reaction include N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), acetonitrile (ACN), dimethylsulfoxide (DMSO) or tetrahydrofuran (THF). The reaction is preferably performed at a temperature of 0-100xc2x0 C. and most preferably at ambient temperature. 
Intermediates of formula IIa or IIb may be prepared from compounds of formula IIIa or IIIb, wherein R1a, R1b, R2a, R2b, R3a, R3b, A1 and B1 are as defined in formula I, by condensation with a compound of formula IVa or IVb, wherein Nxe2x95x90Cxe2x95x90O is an isocyanate group and La or Lb are suitable leaving groups (or synthetic precursors) representing halogen, triflate (TfO), methanesulfonate (MsO), or p-toluenesulfonate (pTsO) and A2, B2, and Z are as defined in formula I, in the presence of a suitable solvent. Suitable solvents for the reaction include N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), acetonitrile (ACN), dimethylsulfoxide (DMSO), tetrahydrofuran (THF), chloroform, ethyl acetate (EtOAc), ethanol (EtOH) or methanol (MeOH). The reaction is preferably performed at a temperature of 0-100xc2x0 C. and most preferably at ambient temperature. 
Compounds of formula IIIa and IIIb are either commercially available or may be prepared by methods known to one skilled in the art (see, for example, xe2x80x98Indoles: Reactions and Synthesisxe2x80x99 in xe2x80x98Heterocyclic Chemistryxe2x80x99, 3rd Edition, J. A. Joule, K. Mills, and G F. Smith, (Pub.) Stanley Thornes Ltd. (1998) and references therein).
Compounds of formula IIIa and IIIb, wherein A1 represents NR4a where together R3a and R4a form a ring and/or wherein B1 represents NR4b where together R3b and R4b form a ring, may be prepared by methods known to one skilled in the art (see, for example, J. E. Macor, J. T. Froman, R. J Post, K. Ryan, Tetrahedron Lett., 38, 1673-1676, 1997).
Compounds of formula IV, IVa and IVb are commercially available or may be prepared by methods known to one skilled in the art.
Where necessary, hydroxy, amino or other reactive groups may be protected using a protecting group as described in the standard text, xe2x80x98Protecting Groups in Organic Synthesisxe2x80x99, 3rd Edition, T. W. Greene and P. G. M. Wuts, 1999, J Wiley and Sons, Inc.
The above described reactions, unless otherwise noted, are usually conducted at a pressure of about one to about three atmospheres, preferably at ambient pressure (about one atmosphere). Unless otherwise stated, the above described reactions are conducted under an inert atmosphere, preferably under a nitrogen atmosphere.
The compounds of the invention and intermediates may be isolated from their reaction mixtures by standard techniques.
Acid addition salts of the compounds of formula I which may be mentioned include salts of mineral acids, for example the hydrochloride and hydrobromide salts; and salts formed with organic acids such as formate, acetate, maleate, benzoate, tartrate, and fumarate salts. Acid addition salts of compounds of formula I may be formed by reacting the free base or a salt, enantiomer or protected derivative thereof, with one or more equivalents of the appropriate acid. The reaction may be carried out in a solvent or medium in which the salt is insoluble or in a solvent in which the salt is soluble, e.g., water, dioxane, ethanol, tetrahydrofuran or diethyl ether, or a mixture of solvents, which may be removed in vacuum or by freeze drying. The reaction may be a metathetical process or it may be carried out on an ion exchange resin. The compounds of formula I exist in tautomeric or enantiomeric forms, all of which are included within the scope of the invention. The various optical isomers may be isolated by separation of a racemic mixture of the compounds using conventional techniques, e.g. fractional crystallization, or chiral HPLC. Alternatively the individual enantiomers may be made by reaction of the appropriate optically active starting materials under reaction conditions which will not cause racemization.
The compounds of formula I, or an enantiomer thereof, and pharmaceutically acceptable salts thereof, may be used on their own or in the form of appropriate medicinal preparations for enteral or parenteral administration. According to a further aspect of the invention, there is provided a pharmaceutical composition including preferably less than 80% and more preferably less than 50% by weight of a compound of the invention in admixture with an inert pharmaceutically acceptable diluent or carrier.
Examples of diluents and carriers are:
for tablets and dragees: lactose, starch, talc, stearic acid; for capsules: tartaric acid or lactose;
for injectable solutions: water, alcohols, glycerin, vegetable oils; for suppositories: natural or hardened oils or waxes.
There is also provided a process for the preparation of such a pharmaceutical composition, which comprises mixing the ingredients.
It will be understood that a pharmaceutical composition comprising a positive modulator of a nicotinic receptor agonist together with a pharmaceutically acceptable carrier said positive modulator having the capability to increase the efficacy of the said receptor agonist. For the purposes of the present invention, the term xe2x80x9cpositive modulatorxe2x80x9d or xe2x80x9cpositive modulator of a nicotinic receptor agonistxe2x80x9d shall be understood as a compound having the capability to increase the maximum efficacy of a nicotinic receptor agonist.
It will be understood that the invention includes compositions comprising either a positive modulator as the only active substance, thus modulating the activity of endogenous nicotinic receptor agonists such as acetylcholine or choline, or a positive modulator in combination with a nicotinic receptor agonist. Thus, the said pharmaceutical compositions containing a positive modulator of a nicotinic receptor agonist may, in addition comprise a nicotinic receptor agonist.
In a preferred form of the invention, the said nicotinic receptor agonist is an xcex17-nicotinic receptor agonist. Example of an xcex17-nicotinic receptor agonist is (xe2x88x92)-Spiro[1-Azabicyclo[2.2.2.]Octane-3,5*-Oxazolidine]-2*-One. Several xcex17-nicotinic receptor agonists are known in the art, e.g. from WO 96/06098, WO 97/30998 and WO 99/03859.
A further aspect of the invention provides a method for the treatment of a condition associated with reduced nicotine transmission, by administering to a patient in need of such treatment, a medically effective amount of a positive modulator of a nicotinic receptor agonist, said positive modulator having the capability to increase the efficacy of the said nicotinic receptor agonist.
It will be understood that the methods of treatment of this invention includes either a positive modulator as the only active substance, thus modulating the activity of endogenous nicotinic receptor agonists such as acetylcholine or choline, or a positive modulator administered together with a nicotinic receptor agonist.
In another preferred form of the invention, the said method of treatment includes a nicotinic receptor agonist, which is an xcex17-nicotinic receptor agonist. Example of an xcex17-nicotinic receptor agonist is (xe2x88x92)-Spiro[1-Azabicyclo[2.2.2.]Octane-3,5 *-Oxazolidine]-2*-One. Several xcex17-nicotinic receptor agonists are known in the art, e.g. from WO 96/06098. WO 97/30998 and WO 99/03859.
A further aspect of the invention is the use of compound according to the invention in the manufacture of a medicament for the treatment or prophylaxis of a condition associated with reduced nicotinic receptor transmission or a condition associated with reduced nicotinic density which could be one of the below mentioned diseases or conditions which comprises administering a therapeutically effective amount of compounds according to the invention to a patient.
It will be understood that the use includes compositions comprising either a positive modulator as the only active substance, thus modulating the activity of endogenous nicotinic receptor agonists such as acetylcholine or choline, or a positive modulator in combination with a nicotinic receptor agonist. Thus, the said use of pharmaceutical compositions containing a positive modulator of a nicotinic receptor agonist may, in addition comprise a nicotinic receptor agonist.
In a preferred form of the invention, the use of the said nicotinic receptor agonist is represented by an xcex17-nicotinic receptor agonist. Example of an xcex17-nicotinic receptor agonist is (xe2x88x92)-Spiro[1-Azabicyclo[2.2.2.]Octane-3,5*-Oxazolidine]-2*-One. Several xcex17-nicotinic receptor agonists are known in the art, e.g. from WO 96/06098, WO 97/30998 and WO 99/03859.
Examples of diseases or conditions include schizophrenia, mania and manic depression, anxiety, Alzheimer""s disease, learning deficit, cognition deficit, attention deficit, memory loss, Lewy Body Dementia, Attention Deficit Hyperactivity Disorder, Parkinson""s disease, Huntington""s disease, Tourette""s syndrome, jetlag, and nicotine addiction (including that resulting from exposure to products containing nicotine).
It will be understood that the said positive modulator can be administered either with the purpose of acting on endogenous nicotine receptor agonists such as acetylcholine or choline, or in combination with an exogenous nicotinic receptor agonist.
A further aspect of the invention relates to a compound for treating or preventing a condition or disorder as exemplified above arising from dysfunction of nicotinic acetylcholine receptor neurotransmission in a mammal, preferably a human, compositions comprising either a positive modulator as the only active substance, thus modulating the activity of endogenous nicotinic receptor agonists, or a positive modulator in combination with a nicotinic receptor agonist. Thus, the said use of pharmaceutical compositions containing a positive modulator of a nicotinic receptor agonist may, in addition comprise a nicotinic receptor agonist, effective in treating or preventing such disorder or condition and an inert pharmaceutically acceptable carrier.
The activity of the compounds of the invention may be measured in the tests set out below:
(a) Xenopus Oocyte Current Recording
The Xenopus oocyte has provided a powerful means of assessing the function of proteins thought to be subunits of ligand-gated ion-channels. Injection of RNA transcribed from cDNA clones encoding the appropriate receptor subunits, or injection of cDNA in which the coding sequence is placed downstream of a promoter, results in the appearance of functional ligand-gated ion-channels on the surface of the oocyte (see e.g. Boulter et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7763-7767).
Consequently, one convenient technique to assess the enhancement of nicotinic efficacy is two-electrode voltage-clamp recording from Xenopus oocytes expressing xcex17-nicotinic receptors from cRNA.
Xenopus laevis frogs (Xenopus I, Kalamazoo, Mich.) were anesthetized using 0.15% tricaine. Oocytes were removed to OR2 solution (82 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 1.5 mM NaH2PO4, 1 mM MgCl2, 0.1 mM EDTA; pH 7.4). The oocytes were defolliculated by incubation in 25 ml OR2 containing 0.2% collagenase 1A (Sigma) two times for 60 min on a platform vibrating at 1 Hz and stored in Leibovitz""s L-15 medium (50 xcexcg/ml gentomycin, 10 Units/ml penicillin, and 10 xcexcg/ml streptomycin). Approximately 50 ng of cRNA was injected in each oocyte the following day. cRNA was synthesised from cDNA using Message Machine (purchased from Abion).
The external recording solution consisted of 90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM BaCl2, 5 mM HEPES; pH 7.4. Two-electrode voltage-clamp recording was carried out using an Oocyte Clamp amplifier (OC 725C; Warner Instrument, Hamden, Conn.). Oocytes were impaled with two electrodes of 1-2 Mxcexa9 tip resistance when filled with 3M KCl. Recordings were begun when membrane potential became stable at potentials negative to xe2x88x9220 mV (resting membrane potentials are less negative when Ba++ replaces Caxe2x88x92+ in bathing solutions). Membrane potential was clamped at xe2x88x9280 mV. ACh was purchased from Sigma. Oocytes were continuously perfused (5 ml/min) with recording solution with or without ACh.
Current amplitude was measured from baseline to peak. EC50 values, maximal effect, and Hill slopes were estimated by fitting the data to the logistic equation using GraphPad Prism (GraphPad Software, Inc., San Diego, Calif.).
Increases in agonist efficacy elicited by a positive modulator can be calculated in two ways:
(1) As percent potentiation of current amplitude which is defined as 100(Im-Ic)/Ic where Im is current amplitude in the presence of modulator and Ic is current in the absence of modulator.
(2) As percent potentiation of xe2x80x9carea under curvexe2x80x9d of an agonist trace, which is the integration of net current over time. Area under the curve is a common representation of the total ion flux through the channel.
(b) Ca2+ Flux Imaging
Imaging of Ca2+ flux through nAChR xcex17 receptors transiently expressed in a cell line is another means of assaying modulator activity.
Cells expressing xcex17 receptors (for example HEK-293 cells or cell cultured neurons) are grown to confluence in 96 well plates and loaded with fluo-3, a fluorescent calcium indicator. To screen for xcex17 modulatory activity, the 96 well plate is placed in a fluorescence imaging plate reader (FLIPR) and test compounds along with an xcex17 agonist are applied simultaneously to all well. Receptor activation is measured by calcium influx into cells which is quantified by the increase in fluorescence intensity of each well, recorded simultaneously by the FLIPR. A modulatory effect is determined by the increase in fluorescence over that of agonist alone. Similarly, to test for nAChR xcex17 agonist activity, test compounds along with an xcex17 modulator are applied simultaneously to all wells. Receptor activation is measured by calcium influx into cells which is quantified by the increase in fluorescence intensity of each well, recorded simultaneously by the FLIPR. An agonist effect is determined by the increase in fluorescence over that of modulator alone. Cell-cultured neurons are prepared according to the following method: Eighteen day old Sprague-Dawley rat fetuses (E-18) were asceptically removed from the pregnant male, sacrificed, the frontal cortices of the brains removed, the menniges stripped, and the cleaned cortex placed into cold HBSS. If hippocampus was desired, the hippocampus was dissected away from the cortex and then placed into cold HBSS. The tissues were mechanically dispersed, washed once in HBSS (200 g for 30 minutes in 4xc2x0 C.) resuspended in a modification of Sato""s medium supplemented with glutamine, antibiotics, potassium chloride, insulin, transferrin, selenium, and 5% heat-inactivated fetal bovine serum (FBS; endotoxin free) and plated into each of a 24-well plate (coated with poly-L-lysine). The wells could contain glass coverslips which were also coated with PLL. The plates were incubated at 37xc2x0 C. in a CO2 incubator. After 24 hours the medium was removed, fresh medium added, and the cells allowed to grow for at least another 11 days, feeding when necessary.
The compounds of the invention are compounds, which causes a 100% potentiation (2-fold increase) of baseline current (as described above), as measured baseline to peak at low concentration of Acetylcholine (30 xcexcM), indicating that they are expected to have useful therapeutic activity. The compounds of the invention are also compounds, which increase the flux of Ca2+ when applied in the Ca2+ flux-imaging assay, as described above. Any increase of Ca2+ flux, caused by a compound of the invention, compared to the Ca2+ flux caused by an agonist alone (as measured in Fluorescence Intensity Units) indicates that they are expected to have useful therapeutic activity.
The use of compounds of the invention have the advantage that they may be less toxic, be more efficacious, be longer acting, have a broader range of activity, be more potent, produce fewer side effects, are more easily absorbed or have other useful pharmacological properties.
Commercial reagents were used without further purification. Mass spectra were recorded following either chemical ionization (MS CI) or electrospray (MS ES) ionization methods and are reported as m/z for the protonated parent molecular ion (MH+). Room temperature refers to 20-25xc2x0 C.